Pros. NatL Acad. Sci. USA Vol. 79, pp. 2064-2067, March 1982 Medical Sciences.

Nonselective expression of simian virus 40 large tumor fragments in mouse cells (recombinant DNA) V. B. REDDY*t,. S. S. TEVETHJAt, M. J. TEVETHIAt, AND S. M. WEISSMAN* *Deparment of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06510; and *Department of Microbiology, The Milton S. Hershey Mei enter, The Pennsylvania State University, Hershey, Pennsylvania 17033 Communicated by Aaron B. Lerner, December 21, 1981

ABSTRACT To understand the role of various functional do- mids containing the virus serotype 1 (HSV-1) mains ofsimian virus 40 early tumor , we have cloned and thymidine kinase (TK) gene linked to fragments of SV40 DNA introduced into mouse cells portions ofearly simian virus 40 DNA. coding for regions of the early into TK-deficient cells Two types of truncated large tumor.antigen (33 and 12.3 kilodal- and selected for cells stably producing thymidine kinase. A tons), as well as smalltumor antigen, were identified by immu- majority of cell clones obtained in this. fashion express the un- noprecipitation. Both truncated large tumor antigens have been selected SV40 peptides encoded by the SV40 DNA fragment. found to be overproduced with respect to the small tumor antigen, Various partial peptides were readily detectable, although they although the 12.3-kilodalton truncated was had different more stable than the 33-kdlodalton one. Nonviral 53-kdlodalton stabilities. These peptides could be used in lo- was not found associatedwitheither truncated large tumor cating antigenic domains in the . antigen in immunoprecipitations. MATERIALS AND METHODS A small plaque isolate ofSV40 strain 776 (the variant with a 72- Simian virus 40 (SV40) encodes two early proteins, small tumor base pair tandem repeat near the origin ofreplication), bacterial (t) antigen and large tumor (T) antigen (1). The T protein is in- plasmid pBR322, and the BamHI restriction fragment of HSV- volved in the onset of viral DNA replication (2), repression of 1, containing the herpes TK gene (16-20), were used as starting early RNA synthesis (3), transformation ofvarious tissue culture materials for DNA in constructions. Restriction endonucleases cells (4), activation of host ribosomal genes (5), stimulation of used were obtained from commercial sources or prepared ac- cellular DNA synthesis (6), adenovirus helper function (7), and cording to published protocols. DNA restriction enzyme digests induction oftumor-specific transplantation antigen (8, 9). Dele- and ligations, bacterial transformations, and sequence analyses tion mutants (10) that do not produce t antigen, but only T an- ofplasmids were carried out according to published procedures. tigen, are capable ofeffecting all these functions (11). Although The nature ofeach construction was confirmed by multiple re- it has been suggested that t antigen may have a helper function striction endonuclease digestions and partial sequence analyses. or act like a hormone (10), its role is not well understood. The cells used were mouse LMTK- cells obtained initially from Conventional transformation ofcells by SV40 is based on the William Summers and S. Silverstein. Restriction endonucleases selection ofcells that exhibit altered growth. This requires suf- and T4 DNA ligase were obtained from commercial sources. ficient viral genetic information or alteration in host-cell gene Construction ofBacterial Plasmids. The plasmids used were expression to produce complex phenotypic changes. Some ex- made following National Institutes of Health containment periments have been carried out to obtain cells that have in- guidelines. Only a brief description of their constructions is corporated intact SV40 DNA without specific selection for the given below. transformed phenotype (12, 13). These experiments were lim- Plasmid containing the entire early region, pVBETK-l. ited by lack ofselection methods for cells bearing viral EcoRI linkers (G-G-G-A-A-T-T-C-C-C) were added to HpaII- andconfined to situations in which intact genes forviral proteins cut nuclease Sl-treated SV40 linear DNA and the DNA was were present. We and others (13, 14) have used selection for restricted with EcoRI/BamHI. The portion of the DNA be- covalently linked genes to obtain cell lines that have acquired tween 0.72 and 0.14 map units (m.u.), containing the entire stably associated SV40 sequences without prior selection for the early region including the SV40 origin, was cloned between the functions of these sequences. EcoRI and BamHI sites of pBR322. The resulting plasmid, Recently, separable functional domains within the early pro- pVBE, was cut with BamHI and the HSV-1 BamHI fragment teins have been identified by microinjecting restriction endo- containing the TK gene was inserted to obtain pVBETK-1. nuclease-generated DNA fiagments of T-antigen-coding seg- Plasmids containing truncated portions ofT antigen and the ments directly or after cloningplasmid pBR322 into mammalian entire t antigen, pVBtTK-1 and pVBtITK-1. The construction cells (15). These studies are limited to functions that can be ofpVBtl is as shown in Fig. 1. pVBtl was restrictedwith BamHI measured at the level ofsingle microinjected cells and for which and the HSV-1 TK BamHI fragment was inserted to obtain plas- transient presence of the protein(s) is sufficient. Establishment mid pVBt1TK-l. Plasmid pVBETK-1 was restricted with Hpa ofcell lines that continuously express truncated forms ofT an- I and the large fragment was self-ligated to obtain pVBtTK-1. tigen would markedly enhance functional analysis of specific Plasmid containing only a short NH2-terminal portion of T segments of this, protein. antigen and the entire t antigen, pVBt2TK-1 . The construction We have examined production of fragments of SV40 early protein in mouse cells. For this purpose, we introduced plas, Abbreviations: SV40, simian virus 40; t and T antigen, small and large tumor antigen, respectively; HSV-1, herpes simplex virus serotype 1; The publication costs ofthis article were defrayed in part by page charge TK, thymidine kinase; m.u., map unit(s); NVT, nonviral T (protein). payment. This article must therefore be hereby marked "advertise- t Present address: Southern Medical and Pharmaceutical Corporation, ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 3500 East Fletcher Avenue, Tampa, FL 33612. 2064 Downloaded by guest on September 27, 2021 Medical Sciences: Reddy et aL Proc. Natl. Acad. Sci. USA 79 (1982) 2065

A Haoe C + Hind m linkers (.091- 194 m u.) (CCAAGCTTGG) Hind m cut R6(.711) I pBR322 B MboanE+dCtails (.517-.599) RI Hindm (I194) I pBR322 PstIl cut and dG tailed BmnHi ( 144) :(.425) RI Mind m I Hind m RI RI Toq pZC550 Hind M (.091) I. Cut with PstI I1. Restrict with RI P5I 2. Si nuclease .711 .649 .566 .425 .330 2. isolate the 1990 BP long fragment 3. Isolate the small -n , BainHI fragment Toq I 1. Cut with Taq I Pull pM9429 - 4 l l 2. Isolate the small RI HindM HindM RI 1. CutwithBamHl .517 .56 .599 fragment 2. Self-ligate the large .711 .649 .425 .330 fragment 1. Cut with Taq I 2. Isolate the large 1. Partial cut with Hind m fragment 2. Isolate the large RI- Hind m RI piece of DNA I Hind m (.194) Ligate, cut with RI RI Hind m Hind m i I pZC265 Hps I (.169) .711 .649 .425 RI Hind E TaqI .711 .6496 .566 .5177 Som HI (.144)

Cut with RI and Hind m 1. Cut with Rl and Hpa I 2. Isolate the large fragment

Ligate Ligate

RI(.711) RI(.7110 Hindm (.649) Hind M (.649)

pV~tI p1/512 Toq I (.566) Hind (.425/ .194) ImFUSion begtwn .517and .169 r.u. B 44) Rn Hi 1 144) FIG. 1. Construction of plasmids pVBtl (A) and pVBt2 (B). Positions of cleavage sites on the SV40 are relative to the unique EcoRI site. -, plasmid DNA; -, SV40 DNA. Hae HI C is the segment of SV40 DNA between 0.20 and 0.08 m.u. of pVBt2 is as shown in Fig. 1B. This plasmid was cut with the SV40 origin sequences (23). Similar observations have been BamHI and TK BamHI DNA was inserted to obtain pVBt2TK- made by others using the CaPO4 method oftransformation (24) 1. or microinjection (25) of TK- cells. Recent evidence indicates Transformation ofLMTK- Cells with Plasmid DNA. Mouse that this may be a consequence of enhanced transcription (24, LMTK-cells were maintained in Dulbecco's modified Eagle's 26). medium/5% fetal calfserum/0.03% glutamine/0.075% NaHCO3 We then examined viral in cells in which the supplemented with penicillin at 100 units/ml and streptomycin whole SV40 early gene region and the SV40 early and at 100 kug/ml. The transformation procedure used was essen- polyadenylation signal were integrated into pBR322 next to tially as described (21), except that the carrier DNA used (final HSV-1 BamHI TK gene fragment (plasmid pVBETK-1). A mass concentration, 10 jug/ml) was isolated from LMTK- cells. The culture of transformed cells and nine individual clones were TKV transformants were selected in Dulbecco's modified Ea- expanded and examined. One clone was totally negative for T gle's medium containing hypoxanthine at 15 Ag/ml aminop- antigen and one showed some T-antigen-negative cells while, terin at 0.2 ,ug/ml, and thymidine at 5 pug/ml. Individual clones in the remaining seven clones, every cell was positive for T an- were picked by using cloning pipettes and grown into mass cul- tigen by immunofluorescence. ture in the same medium. Lines ofcloned cells were labeled in vitro with [S3]methionine Preparation ofCell Extracts, Immunoprecipitation, and Gel and SV40 T and t antigens were immunoprecipitated and ex- Electrophoresis. Transformed LMTK' cells were grown in hy- amined by electrophoresis on NaDodSOjpolyacrylamide gels. poxanthine/aminopterin/thymidine medium and radiolabeled As shown in Fig. 2, these cells synthesized normal-size SV40 with [35S]methionine. Cell extracts were prepared and proteins T and t antigens, as well as the 55-kilodalton (kDal) cellular were immunoprecipitated with normal hamster serum or serum nonviral T (NVT) protein (Fig. 2), which has been shown to as- from hamsters bearing tumors induced by SV40-transformed sociate with T antigen (27-30). cells and analyzed by using polyacrylamide gel electrophoresis Cells transformed by pVBtTK-1, which contains the SV40 as described (22). early region deleted between 0.37 and 0.17 m.u., produced t antigen but no trace of the expected 40-kDal truncated T an- RESULTS tigen (Fig. 3A). We have also observed that the NVT protein was not immunoprecipitated by antiserum against viral T an- During the course of transformation of LMTK- cells, we ob- tigen. We do not know the reason for the absence of truncated served that TK plasmids (pVBETK, pVBtlTK, pVBt2TK) con- T protein.in the cells transformed by pVBtTK-1. It may be that verted LMTK- cells into TK cells more efficiently than did the T-antigen splice is not occurring properly or that the protein the HSV-1 TK plasmid alone, containing no SV40 sequences. itself is unstable. A 10- to 30-fold increase in the number oftransformed colonies Cells transformed by pVBtlTK-1, the plasmid containing the was seen when plasmids contained the SV40 origin with or with- first third of the SV40 early region (Fig. 3B), did not show T out T-antigen-coding regions. The increased TK transformation antigen by immunofluorescence and did not demonstrate either was independent of the orientation of the TK gene relative to normal-sized T antigen or NVT protein but showed t antigen Downloaded by guest on September 27, 2021 2066 Medical Sciences: Reddy et aLPProc. Nad Acad. Sci. USA 79 (1982)

1 2 34 5 6 7 8 9101112 kDal 1 2 3 4 5 6 7 8 9 10 11 12 kDaI

wm .. _

Toe 40 *0 --O - -92.5 -- 92.5 -69 - 69 NVT-' _.g -46 - 46

-30

- 30

t-

-12.3

FIG. 2. Synthesis and stability of SV40 T, t, and NVT antigens in pVBETK-1-transformed LM[TK' cells. Cultures were pulse labeled for 30 min with [35S]methionine (50 ACi/ml; 1 Ci = 3.7 x 1010 becquerels) TS S _. - 12.3 in methionine-free medium (lanes 1 and 2) and radioactive label was chased in the presence of a 100-fold excess of unlabeled methionine for 30 min (lanes 3 and 4), 1 hr (lanes 5 and 6), 2 hr (lanes 7 and 8), 4 hr FIG. 4. Stability of t and truncated T antigens in pVBt2TK-1- (lanes 9 and 10), and 20 hr (lanes 11 and 12). Extracts were immu- transformed LMTK' cells. Cultures were pulse labeled with noprecipitated with antitumor serum (lanes 1, 3, 5, 7, 9, and 11) or [35S]methionine (50 ,uCi/ml; lanes 1 and 2) and chased in the presence normal hamster serum (lanes 2, 4, 6, 8, 10, and 12) and analyzed by of a 100-fold excess of unlabeled methionine for 20 min (lanes 3 and NaDodSO4/polyacrylamide gel electrophoresis. The band at the top is 4), 40 min (lanes 5 and 6), 1 hr (lanes 7 and 8), 2 hr (lanes 9 and aggregated material that did not enter the gel. 10), and 4 hr (lanes 11 and 12). Extracts were immunoprecipitated with antitumor serum (lanes 2, 4, 6, 8, 10, and 12) or normal hamster serum and a 33-kDal T-antigen-related peptide similar to that pro- (lanes 1, 3, 5, 7, 9, and 11) and analyzed by NaDodS04Jpolyacrylamide duced by a nonviable deletion mutant of SV40 virus (31). Cells gel electrophoresis. The doublet seen for the 12.3-kDal T protein (T*) transformed by plasmid pVBt2TK-1, which contains only the may be a gel artifact because a single band is observed in many other coding region for t antigen and early splice and polyadenylation immunoprecipitations. sites ofSV40, synthesized a normal-sized t antigen andapeptide of =13,000 kDal (Fig. 4). Also, the immunoprecipitates from cells transformed by pVBt2TK-1 did not show any NVT protein and the cells showed no T-antigen immunofluorescence. A 1 2 B The identity of the 33-kDal protein of pVBtlTK-1 to au- thentic truncated T antigen was confirmed by the fact that it was not precipitated by an antitumor serum that is specific for t an- 1 2 3 4 5 6 7 8 9 10 kDal tigen (22). In addition, the truncated.T antigen was not precip- itated by antitumor serum specific for the carboxy terminus of -92.5 T antigen. Antiserum prepared against denatured T antigen (32-34) did react with truncated T antigen and, as shown pre- -69 viously, with t antigen as well (data not shown). Truncated T -46 antigen synthesized by pVBtLTK-1-transformed cells is similar in size to the 33-kDal truncated T antigen synthesized by cells T- infected by the.SV40 deletion mutant dl 1001 (31) and the 33- -30 to 37-kDal truncated T reported here is phosphorylated (data not shown), as was the 33-kDal T antigen synthesized in dl 1001-infected cells. t - _ To assess the stability ofthe truncated T antigens, cells were labeled with [NS]methionine and chased with excess unlabeled 46 a-*- t -12.3 methionine for various times before immunoprecipitation (Figs. 3 and 4). Methionine radioactivity disappeared relatively rap- idly from the 33-kDal truncated T antigen produced by pVBtlTK-1 while the 12-kDal truncated T antigen produced by FIG. 3. Synthesis and stability of truncated T and t antigens in pVB2TK-1 was relatively stable over the period of the chase, pVBtlTK-1- and pVBtTK-1-transformed LMTK' cells. (A) Cultures as was intact wild-type T antigen (Figs. 2, 3, and 4). In view of of pVBtTK-1-transformed cells were labeled in Fig. 2 for 20 min but the small number of methionine residues in these truncated T not chased, and the extracts were immunoprecipitated with normal hamster serum (lane 1) or antitumor serum (lane 2) and analyzed by antigens, both the 12- and the 33-kDal truncated T antigens NaDodSO4/polyacrylamide gel electrophoresis. (B) Cultures of seem to be produced in larger amounts than intact T antigen. pVBtlTK-1-transformed cells were pulse labeled for 1 hr with This was particularly marked in the case of the 12-kDal trun- [35Slmethionine (50 ,uCi/ml; lanes 1 and 2) and chased in the presence cated T antigen for which a relatively low amount oft antigen of a 100-fold excess of unlabeled methionine for 20 min (lanes 3 and was produced. Untransformed cells and cells transformed by 4), 40 min (lanes 5 and 6), 1 hr (lanes 7 and 8), and 2 hr (lanes 9 and the various plasmids produced similar amounts ofNVT protein 10). Extracts were immunoprecipitated with antitumor serum (lanes 2, 4, 6, 8, and 10) or normal hamster serum (lanes 1, 3, 5, 7, and 9) and after a brief pulse-labeling period as detected by immunopre- analyzed by NaDodS04/polyacrylamide gel electrophoresis. Doubling cipitation with monoclonal antibody against NVT protein of the truncated T antigen (T*) was an inconsistent finding. (kindly provided by E. Harlow), in agreement with the evidence Downloaded by guest on September 27, 2021 Medical Sciences: Reddy et al. Proc. Natl. Acad. Sci. USA 79 (1982) 2067 (34) that the accumulation of NVT protein in SV40-transformed 2. Tegtmeyer, P. (1975)J. Virol 15, 613-618. cells is due to stabilization rather than accelerated synthesis of 3. Tegtmeyer, P., Schwartz, M., Collins, J. K. & Rundell, K. (1975) J. Virol 16, 168-178. the protein. 4. Martin, R. G. & Chou, J. Y. (1975)J. Virol. 15, 599-612. 5. Soprano, K. J., Dev, V. J., Croce, C. M. & Baserga, R. (1980) DISCUSSION Proc. Natl Acad. Sci. USA 76, 3885-3889. Our results show that it is possible to nonselectively introduce 6. Henry, P., Black, P. H., Oxman, M. N. & Weissman, S. M. into cells gene fragments coding for truncated or incomplete (1966) Proc. Natl Acad. Sci. USA 56, 1170-1176. proteins and to obtain cell lines that stably produce high levels 7. Cole, C. N., Crawford, L. V. & Berg, P. (1979) J. Virol 30, of the proteins. Other workers have noted an apparent epige- 683-691. 8. Chang, C., Martin, R. G., Livingston, D. M., Luborsky, S. W., netic phenomenon in which cotransfected SV40 viral early re- Hu, C. & Mora, P. T. (1979) J. Virol 29, 69-75. gions are not stably expressed in all cells containing the viral 9. Tevethia, S. S., Flyer, D. C. & Tjian, R. (1980) Virology 10, gene (3, 5). In the present series of results, the representative 13-23. clones analyzed from each transformation experiment contin- 10. Shenk, T. E., Carbon, J. & Berg, P. (1976)J. Virol. 18, 664-671. ued to express the viral proteins for >25-44 passages, as shown 11. Lebowitz, P. & Weissman, S. M. (1979) Curr. Top. Microbiot by immunoprecipitation and NaDodSOjpolyacrylamide gel Immunol 87, 86-87. 12. Risser, R. & Pollock, R. (1974) Virology 59, 477-489. electrophoresis. However, only cells transformed with the in- 13. Linnenbach, A., Huebner, K. & Croce, C. M. (1980) Proc. Natl tact early region of SV40 were examined for immunofluores- Acad. Sci. USA 77, 4875-4879. cence, because the smaller peptides did not give satisfactory 14. Hanahan, D., Lane, D., Lipsich, L., Wigler, M. & Botchan, M. immunofluorescence with the available antisera. In those (1980) Cell 21, 127-139. clones expressing a high level of T antigen, every cell showed 15. Galanti, N., Jonas, G. J., Soprano, J., Floros, J., Weissman, S. typical nuclear immunofluorescence. We do not know whether M., Reddy, V. B., Tilighman, S. & Baserga, R. (1981) J. Biol Chem. 256, 6469-6474. the lack ofexpression of SV40 T antigen in a minority ofclones 16. McKnight, S. L. & Croce, C. M. (1979) Carnegie Inst. Yearb. 98, is a result ofDNA rearrangement or an epigenetic phenomenon 56-61. of the type previously described (14). 17. Wigler, M., Silverstein, S., Lee, L.-S., Pellicer, A., Cheng, Y- The stability of the T-antigen fragments was not simply re- C. & Axel, R. (1977) Cell 11, 223-232. lated to their size and may prove to be a more complex function 18. Colbere-Garapin, F., Chousterman, S., Horodniceanu, F., of the amino acid sequences and potential protein structures Kourilsky, P. & Garapin, A.-C. (1979) Proc. Natl Acad. Sri. USA 76, 3755-3759. formed by various partial peptides. It is curious to observe that 19. Wilkie, N. M., Clements, J. B., Boll, W., Mantei, N., Lonsdale, plasmid pVBtITK-1, which produces an unstable 33-kDal pro- D. & Weissman, C. (1979) Nucleic Acids Res. 7, 859-877. tein, has its deletion near the region specifying temperature 20. Enquist, L. W., Van de Woude, G. F., Wagner, M., Smiley, J. sensitivity to the T antigen. R. & Summers, W. C. (1979) Gene 7, 335-342. The present approach makes it possible to obtain mass cul- 21. Wigler, M., Pellicer, A., Silverstein, S., Axel, R., Uslaub, G. & tures of cells overproducing the protein fragments, either for Chasin, L. (1979) Proc. NatI Acad. Sci. USA 76, 1373-1376. 22. Greenfield, R. S., Flyer, D. C. & Tevethia, S. S. (1980) Virology isolation of the fragments or for study of their effects in trans- 104, 312-322. formed cells. For example, cells producing the 33-kDal trun- 23. Reddy, V. B. & Weissman, S. M. (1980) SV40, Polyoma and Ad- cated form ofT antigen express a SV40-specific transplantation- enoviruses (Cold Spring Harbor Laboratory, Cold Spring Harbor, rejection antigen at the cell surface that can be detected by the NY), p. 109. lymphocyte mediated cytotoxicity assay (data not shown). Plas- 24. Schaffner, W. (1981) Cell, 27, 299-308. mid pVBtTK-1 should be useful in studying the biological role 25. Capecchi, M. R. (1980) Cell 22, 479-488. 26. Chambon, P. (1981) Nucleic Acids Res. 9, 6047-6068. of t antigen. By using transformants generated by the meth- 27. Linzer, D. H. & Levine, A. J. (1979) Cell 17, 43-52. odology described here, it should be possible to precisely define 28. Lane, D. P. & Crawford, L. V. (1979) Nature (London) 278, regions of SV40 T antigens that act as virus-specific transplan- 261-263. tation-rejection antigen. In addition, localization ofsuch aspects 29. McCormick, F., Clark, R., Harlow, E. & Tjian, R. (1981) Nature ofT-antigen functions as DNA and NVT protein binding could (London) 292, 63-65. be investigated. 30. Harlow, E., Pim, D. C. & Crawford, L. V. (1981) J. Virol 34, 752-763. This investigation was supported by Grants CA 25000 (to S. S.T.), 31. Rundell, K., Collins, J. K., Tegtmeyer, P., Ozer, H. L., Lai, C.- CA24694 (to M.J.T.), and CA 16038 (to V.B.R. and S.M.W.) from the J. & Nathans, D. (1977)J. Virol 21, 636-646. National Cancer Institute. 32. Lane, D. P. & Robbins, A. K. (1978) Virology 87, 182-193. 1. Tooze, J., ed. (1980) The Molecular Biology of Tumor Viruses, 33. Lanford, R. E. & Butel, J. (1979) Virology 97, 296-300. DNA Viruses (Cold Spring Harbor Laboratory, Cold Spring Har- 34. Oren, M., Maltzman, N. & Levine, A. J. (1981) Mol. Cell Biol bor, NY), pt. 1. 1, 101-110. Downloaded by guest on September 27, 2021